Humans survive by maintaining equilibrium with their environment (1). A perturbation to this equilibrium can occur anywhere on the spectrum from minor to severe. The character of the disturbances also fluctuates dramatically with such examples as burns, trauma, sepsis, and surgery. The stress response must be tightly attuned to the severity and character of the insult to restore equilibrium. One of the key players in this complex orchestration of the stress response is the glucocorticoid receptor (GR). The GR is expressed in virtually all cells and is important for metabolism, development, and immune regulation (2, 3). The stress response activates the hypothalamic-pituitary-adrenal axis to produce cortisol that then binds to the GR and alters the production of inflammatory mediators.
The human GR (hGR) is mainly thought to carry out its function by binding cortisol in the cytoplasm, shedding its chaperones, dimerizing, and then translocating to the nucleus where it directly binds to promoter regions and activates transcription of certain genes (2). This transcriptional event culminates in an attenuation of the inflammatory response (3). The hGR gene is located on chromosome 5q31-32 and is reported to consist of nine exons, of which exons 2 through 9 are translated. Alternative splicing of exon 9 produces two isoforms: hGRα (777 amino acids) and hGRβ (742 amino acids) (4). Human GRα is considered the dominant active isoform, whereas hGRβ has been implicated as an inhibitor of hGRα (5). The hGRα protein has been reported to contain four domains, including a transactivation domain, DNA-binding domain (DBD), hinge region, and a ligand-binding domain (LBD) (6).
Despite the stress response being critical to reestablishing this dynamic equilibrium, it is quite variable among individuals. For example, siblings of similar age, although suffering the same total body surface area burn, can have dramatically different outcomes. Similar examples of this variability can be found in patients experiencing trauma or sepsis. In addition, steroid resistance and sensitivity have been noted in both healthy individuals and patients with inflammatory diseases such as inflammatory bowel disease, asthma, and rheumatoid arthritis (7–13). The reason for this variability in the response to steroids has been postulated to result from alternative splicing, posttranslational modifications, alternative translation initiation, and single-nucleotide polymorphisms (SNPs) of the hGR (14–17). Indeed, we have shown that an alternative splice isoform and SNPs can impact the steroid response of the hGR in vitro (14, 18, 19). To better understand the variability of the hGR and ultimately its role in the stress response, we examined variability in the structure and function of the hGR in healthy human volunteers. During our functional analyses of multiple mutations, we identified two naturally occurring hGR SNPs that induced hyperactivity and examined how hGR isoforms containing these SNPs responded to variations in steroid treatment.
MATERIALS AND METHODS
The two SNPs were identified from a pool of 97 volunteer human subjects (70 females and 27 males) that was analyzed to find hGR polymorphisms. Subjects with a history of chronic illness (hypertension, diabetes mellitus, chronic obstructive pulmonary disease, inflammatory bowel disease, autoimmune disease, and cancer), pregnant women, and individuals taking exogenous steroids were excluded from this study. Demographics were previously published (19). All protocols involving the collection, processing, and analysis of human blood samples in this study were approved by the institutional review board of the University of California, Davis. Written informed consent was obtained from all participants in this study.
Identification, construction, and nomenclature of hGR SNPs
Total RNA was isolated from buffy coat samples using the RNeasy Miniprep Kit (Qiagen, Valencia, Calif) with a modified protocol. Reverse transcription polymerase chain reaction using the Sensiscript RT Kit and Taq polymerase (Qiagen) was performed to amplify the hGR coding sequence in two sections (exons 2–3, forward 5′-tcactgatggactccaaag-3′, reverse 5′-aagcttcatcagagcacacc-3′; exon 3-9α, forward 5′-ccagcatgagaccagatgta-3′, reverse 5′-ttaaggcagtcacttttgatgaaac-3′). The fragments were cloned into the pGEM-T Easy vector (Promega, Madison, Wis) and sequenced at MCLAB (South San Francisco, Calif). Polymorphisms were identified by comparison with the hGRα reference sequence (NM_001018077) from the National Center for Biotechnology Informatics (NCBI).
Two SNPs were then chosen for subsequent isolation and analysis. One novel SNP, T1463C, was recently discovered and the other (A2297G) was previously identified by our laboratory (19). Of the 97 healthy subjects, two subjects had the T1463C SNP and one subject had the A2297G SNP. The fragment isolating the SNP was then cut with restriction enzymes for recombination into a full-length coding sequence before ligation into the pcDNA4-HisMax vector (Invitrogen, Carlsbad, Calif) for functional analysis. For a negative control, full-length hGRα was cloned into the pcDNA4-HisMax vector (Invitrogen) in reverse orientation.
Measurement of transactivation potentials of hGR isoforms
tsA201 cells, an HEK 293 cell subclone, were transfected with either plasmids containing individual hGR isoforms (hGRα-NCBI, hGR-T1463C, hGR-A2297G, or reverse hGRα as a negative control). For each transfection experiment, 12,000 cells were seeded in 100 μL of antibiotic-free Dulbecco modified Eagle medium (Invitrogen) with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, Ga) in a 96-well plate and then incubated at 37°C in a 5% CO2 atmosphere. The day after seeding, the cells were transfected with the appropriate hGR isoforms and a glucocorticoid response element–luciferase reporter plasmid (PathDetect GRE Cis-Reporter Plasmid; Agilent Technologies, La Jolla, Calif) using Fugene 6 (Roche, Indianapolis, Ind) per the manufacturer’s protocol. Twenty-four hours after transfection, the cells were either assessed for transactivation potential or treated for an additional day with graded concentrations of hydrocortisone (10−4 – 1 μM), methylprednisolone (10−6 – 10−2 μM), dexamethasone (10−7 – 10−3 μM), or vehicle before assessment. The vehicle for hydrocortisone and methylprednisolone was 0.9% saline, and the vehicle for dexamethasone was a solution consisting of 1% benzyl alcohol, 1.1% sodium citrate, and 0.1% sodium sulfite in water. The concentration ranges were determined based on previous titration studies to optimize the response of hGR to each steroid (data not shown). Pharmaceutical-grade hydrocortisone sodium succinate (Pfizer, New York, NY; clinical anti-inflammatory adult dosage, 15–240 mg; half-life, 8–12 h), methylprednisolone sodium succinate (Pfizer; clinical anti-inflammatory adult dosage, 10 – 40 mg; half-life, 12 – 36 h), and dexamethasone sodium phosphate (Luitpold Pharmaceuticals, Shirley, NY; clinical anti-inflammatory adult dosage, 0.4 – 6 mg; half-life, 36 – 72 h) were used. Transactivation potential was assessed using the Luciferase Assay Kit (Agilent Technologies). The luminescence was measured on a Perkin-Elmer MicroBeta Trilux (Perkin-Elmer, Waltham, Mass).
Western blot analysis of hGR isoforms
tsA201 cells transfected with recombinant hGR isoforms were lysed in ice-cold buffer (1% Igepal CA-630, 0.15 M NaCl, 0.01 M Na3PO4 [pH 7.2], 2 mM EDTA, 50 mM NaF, 0.2 mM Na3VO3, and 1 μg/mL aprotinin), and the supernatants harvested. Extracted protein was run on a 7.5% Criterion gel (BioRad, Hercules, Calif). Separated proteins were transferred to a PVDF Hybond-P membrane (GE Healthcare, Piscataway, NJ). Membranes were blocked with 5% nonfat dry milk, washed, and incubated overnight with GR (D8H2) XP rabbit monoclonal antibody (Cell Signaling, Danvers, Mass) in 5% nonfat milk. A secondary anti-rabbit antibody linked to horseradish peroxidase was used for protein visualization via chemiluminescence using the ECL Plus Western Blot Detection System (GE Healthcare).
All experiment samples were run in triplicate, except for the vehicle treatments that were run in duplicate, and each experiment was repeated three times. After multiple experiments confirmed the data patterns, three experiments were combined for presentation of figures and statistics. Values are expressed as means, with error bars representing standard error of the mean. These results were compared by one-way analysis of variance, and significance was confirmed with a Tukey post hoc test.
Initial identification of hGR SNPs
A multitude of SNPs were identified in our study population, and two SNPs were selected for further functional analysis. The T1463C SNP is a previously unreported missense mutation that occurs in exon 4 that codes for proline instead of leucine at codon 488 (L488P) (Fig. 1). T1463C occurs within the hinge region of the hGR and just neighbors the DBD. The second SNP, A2297G, is also a missense mutation that occurs in exon 9 and codes for serine instead of asparagine at codon 766 (N766S). A2297G arises within the LBD, and its hyperactivity had been partially described previously (19). Cells containing the transfected hGR SNPs were tested for activity.
Baseline transactivation potentials of hGRα, T1463C, and A2297G isoforms
In the absence of exogenous steroids, in vitro functional assay transactivation potential of A2297G (P < 0.01) was significantly higher and T1463C (P < 0.05) significantly lower when compared with hGRα (Fig. 2). The negative control, hGRα in the reverse orientation, had negligible activity. Protein expression of the expected size (97 kDa) for reference hGRα was confirmed by Western blot for all of the isoforms (data not shown).
Differential activities of hGRα, T1463C, and A2297G in response to hydrocortisone
We next sought to test the response of the various isoforms to a range of concentrations of hydrocortisone. Treatment with the lowest concentration of hydrocortisone (10−4 μM) revealed that A2297G had significantly greater activity (P < 0.01) in comparison with hGRα, although all activity levels were low. The activity of T1463C, however, was significantly lower (P < 0.05) relative to the reference (Fig. 3). At higher concentrations of hydrocortisone, the overall activity of the reference and two SNP isoforms increased, although the A2297G SNP isoforms maintained significantly higher (P < 0.01) transactivation potentials than hGRα. In contrast, the activity of T1463C increased at these greater hydrocortisone concentrations and was also significantly higher (P < 0.01) than reference hGRα. At the highest concentration (1 μM) of hydrocortisone, the activity of T1463C increased to a point where it was significantly more active (P < 0.01) than A2297G. The negative control was low at all hydrocortisone concentrations tested, although some activity, because of the endogeneous hGRα in the cells, was detected at 1 μM.
Differential activities of hGRα, T1463C, and A2297G in response to methylprednisolone
Next, we stimulated transfected cells with various concentrations of another steroid, methylprednisolone. At the two lower concentrations, the activity of A2297G was significantly greater (P < 0.01) than hGRα (Fig. 4). Overall activity did not markedly increase until cells were treated with 10−2 μM of methylprednisolone; at that concentration, only the T1463C isoform had significantly greater activity (P < 0.01) relative to both the reference and A2297G.
Differential activities of hGRα, T1463C, and A2297G in response to dexamethasone
We compared the transactivation of the hGR isoforms with a final steroid, dexamethasone. Although the concentrations of dexamethasone tested differed because of glucocorticoid potency, the pattern was very similar to that of methylprednisolone, with low activity but significantly greater A2297G activity relative to hGRα at two smallest concentrations and significantly greater T1463C activity (relative to the reference) at the highest concentration tested (Fig. 5). The only major difference was that, at 10−7 μM of dexamethasone, the T1463C activity was significantly less compared with hGRα.
In this study, we have demonstrated that individual SNPs in the hGR can cause wide variations in constitutive activity and response to steroid type and dosage. Interestingly, the isoform containing the novel T1463C SNP (located within the hinge region), was hyperresponsive to higher concentrations of hydrocortisone, methylprednisolone, and dexamethasone. Although the other domains of the hGR (i.e., transactivation domain, DBD, and LBD) have been well studied in terms of their functional roles in transactivation activity, less is known regarding the functional role of the hinge region of this receptor. There are no crystallographic structural data for the hGR hinge region. Although the T1463C hGR SNP has not been studied previously, earlier investigations noted that a three–amino acid insertion at the same codon (488) reduced the transcriptional activation of hGRα to 15% while still retaining the ability to bind dexamethasone (20). In our study, the T1463C isoform had significantly less baseline transcription activity relative to reference hGRα, although the decrease was not as dramatic.
There were significant differences when an isoform containing the T1463C SNP was stimulated with specific concentrations of steroids. The mechanism of how this T1463C SNP elicits this differential response to steroids, especially at various concentrations, is unknown at this point. We do know from prior studies that the hinge region has been shown to be acetylated on lysine residues 494 and 495 (21). This acetylation of GR is important for regulating both its own transcriptional activity and repression of NF-κB (21, 22). We can speculate that the T1463C SNP presents these lysine residues in such a fashion so that they may be more or less likely to become acetylated, depending on whether the cell is in a stressed or unstressed state. In fact, a proline substitution, such as that caused by the T1463C SNP, can introduce a kink into the alpha helix protein structure, making the resulting protein more rigid than when a leucine is at this location (23).
In contrast, the isoform containing the A2297G SNP, located within the LBD, had a significantly higher constitutive activity than hGRα (∼3×) and the T1463C (∼5×) isoforms. We expected that there should be a parallel increase in activity when stimulated with steroids compared with the other isoforms; we were surprised that there was not an analogous increase in activity for the A2297G isoform relative to reference at the highest concentrations of methylprednisolone (10−2 μM) and dexamethasone (10−3 μM). The A2297G SNP is located within the τ2 (second transactivation activity site), and this serine substitution seems to elicit a higher transactivation activity, although the exact mechanism of action remains elusive. Perhaps this new serine is a site of phosphorylation. Indeed, phosphorylation of the hGR has been shown to alter its transcriptional activity (24). We could also speculate that the A2297G SNP might introduce a perturbation in the interaction with heat shock protein chaperones and thus allow the receptor to more easily dimerize and translocate to the nucleus with subsequent transcriptional activation.
Regardless of the mechanisms of both the T1463C and A2297G SNPs, their potential influence on the stress response is clear. The T1463C isoform displays low activity until a certain threshold of steroid concentration is met and then becomes significantly more active than hGRα and A2297G. A future survey using a significantly larger diverse population pool to determine the population frequencies of these SNPs may prove of vital importance. In light of our findings in vitro, we can speculate that, in vivo, an individual harboring the T1463C SNP would respond only at a severe level of injury/stress with high transcriptional activity. In other words, this SNP would only be “turned on” at higher levels of steroids such as during periods of high stress or by medical intervention with methylprednisolone or dexamethasone treatment. It is also interesting to note that the A2297G SNP, which has a high baseline transcriptional activation, was found in a healthy human population. We would expect that an individual with this glucocorticoid-sensitive isoform might demonstrate some of the stigmata of hypercortisolism such as hypertension or diabetes mellitus. Perhaps this individual might be more prone to developing one of these conditions across time. Subsequent experiments to test the activity of these isoforms in response to stressors, such as lipopolysaccharide, may shed further light on the issue.
Previous studies have targeted the discovery of hGR SNPs in disease states or cell lines. Interestingly, we have identified a wide diversity in hGR-SNP isoforms in a healthy human population that also demonstrated a large variation in baseline and steroid-stimulated activity. Our findings indicate that different naturally occurring SNPs (such as T1463C or A2297G) may lead to distinct individualized patient responses, depending on type and dosage of steroid. With the widespread use of steroid therapy in diseases of inflammation such as rheumatoid arthritis, asthma, inflammatory bowel disease, and systemic lupus erythematosus, this information becomes very important. With a larger catalog of hGR isoforms and hGR profiling of patients, one could potentially customize an optimal therapy for a particular individual.
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